*2.9. Immunofluorescence Assays*

Immunofluorescence assays of log-phase promastigotes, heat-shocked promastigotes and axenic amastigotes were performed as described previously [38]. Briefly, 2 × 10<sup>5</sup> cells were washed with 1×PBS and applied on microscopic slides and fixed with ice-cold methanol. Following permeabilization and blocking, the cells were stained with primary anti-HA IgG antibody (polyclonal, mouse, 1:3000; Invitrogen, Carlsbad, CA, USA) and secondary anti-mouse Alexa Fluor® 594 IgG (polyclonal, goat, 1:1000; Thermo Fisher Scientific, Waltham, MA, USA) and DAPI (1:50; Sigma Aldrich, Munich, Germany). Fluorescence microscopy was carried out on an EVOS® FL Auto Cell Imaging System using a 64× magnification.

#### *2.10. In Silico Procedures*

In silico construction of plasmids, DNA and protein sequence analyses was performed using the MacVector software, version 17 (MacVector Inc., Cambridge, UK). Microscopy images were processed using Adobe Photoshop CS3 (Adobe Corp., San Jose, CA, USA) and juxtaposed using Intaglio (Version 3.9, Purgatory Design, Durango, CO, USA). Multi-panel figures were also assembled using the Intaglio software.

In silico design of *SUMO*- and *SENP*-specific sgRNAs and primers for the amplification of the donor repair cassettes was performed using the LeishGEdit online tool [39]. Oligonucleotides were purchased from Sigma-Aldrich (München, Germany).

Gene annotations and reference genomes (version 42) of *L. donovani* BPK were downloaded from the TriTrypDB server. Reads were aligned to the reference genomes using the MacVector software version 17 and Bowtie2 algorithm [40].

Statistical analyses were performed using Prism (version 8, GraphPad Software, San Diego, CA, USA). Ranking tests were performed using the U-test [41]. Differences were considered significant at a level of *p* < 0.05.
